The proposed research is a continued study of mobile group II introns, focusing on group II intron-encoded and related reverse transcriptases (RTs), their biochemical properties, biological functions, evolutionary relation- ships, and biotechnological applications. Mobile group II introns are bacterial mobile genetic elements (re- trotransposons) thought to be evolutionary ancestors of the spliceosomal introns and retroelements of higher organisms. They consist of an autocatalytic intron RNA (a ?ribozyme?) and an intron-encoded RT, which func- tion together to promote RNA splicing and intron mobility to new DNA sites by a process called ?retrohoming?. Retrohoming occurs by a ribozyme-based DNA integration mechanism in which the excised intron RNA re- verse splices directly into a DNA site and is reverse transcribed by the intron-encoded RT, yielding an intron cDNA integrated into the host genome. Mobile group II introns or their close relatives are believed to have used this mechanism to proliferate within the nuclear genomes of ancestral eukaryotes before evolving into spliceosomal introns. Group II intron RTs differ from retroviral RTs structurally and biochemically, and they function not only in intron mobility but also in RNA splicing. In some cases, group II intron RTs have lost mobili- ty functions and evolved into cellular splicing factors, a prominent example being the core spliceosomal protein Prp8. Group II intron-like RTs are also associated with some CRISPR systems, including as RT-Cas1 fusion proteins, which we recently found have the unique ability to integrate both DNA and RNA spacers into CRISPR repeats. Finally, we have adapted mobile group II introns for biotechnological applications, first as gene target- ing vectors (?targetrons?), and more recently as a source of new thermostable RTs (TGIRTs), whose novel properties enable new methods for non-coding RNA analysis and next-generation RNA sequencing (RNA-seq), with potential applications in RNA-based diagnostics. Specific aims of this competitive renewal are: (1) to in- vestigate the distinctive enzymatic properties of group II intron RTs and use directed-evolution approaches to analyze and modify these properties; (2) to investigate how group II intron RTs interact with group II intron RNAs to promote RNA splicing and intron mobility, focusing on structural approaches; (3) to investigate mech- anisms and structures of group II intron-like RTs associated with CRISPR systems; (4) to further develop TGIRT-seq and use it to investigate the origin and function of exosomal and plasma RNAs, impacting RNA- based diagnostics for human diseases.